Method for preparing novel cyclomaltodextrin gluccanotransferase

ABSTRACT

A novel cyclomaltodextrin transferase (EC. 2, 4, 1, 19) which preferentially produces β-cyclodextrin from starch or partial hydrolysate thereof and produces a carbohydrate containing β-cyclodextrin as the main components, and a method for preparation thereof comprising cultivating a cyclomaltodextrin glucanotransferase producing microorganism belonging to a moderate thermophile B. coagulans are provided.

This is a division, of application Ser. No. 07/305,631, filed on Feb. 3,1989.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel cyclomaltodextrin transferase(EC. 2, 4, 1, 19; hereinafter referred to as CGTase) which preferentialyproduces betacyclodextrin (hereinafter referred to as β-CD) from starchand a method for preparation thereof. The present invention particularlyrelates to a novel CGTase which is obtained by cultivation of Bacilluscoagulans and which preferentially produces β-CD from starch or partialhydrolysates thereof to form a carbohydrate containing β-CD as maincomponents, and a method for preparation thereof.

2. Description of the Prior Art

A CGTase is an enzyme catalyzing the following reactions: CD producingreactions producing cyclodextrins (hereinafter referred to as CDs) byacting on the α-1,4-glucopyranoside bond of an α-1,4-glucan such asstarch, amylose, amylopectin and glycogen or partial hydrolysatethereof; coupling reactions cleaving CDs and transferring their glycosylresidues to several glycosyl acceptors such as sucrose andmaltooligosaccharides; and disproportionation reactions producingmaltooligosaccharides with various molecular weights by intermoleculartransferring reaction of various maltooligosaccharides. α-CD(cyclohexaamylose) consisting of six glucose units, β-CD(cycloheptaamylose) consisting of seven glucose units and Γ-CD(cyclooctaamylose) consisting of eight glucose units are well-known asCDs but branched CDs branching at the 6th-position of glucose of CDsthrough α-1,6-glucopyranoside bond are also manufactured.

These CDs have a character to make inclusion complexes by incorporatingvarious organic compounds (referred to as guest compounds) into cavitiesthereof due to their specific structure exhibiting hydrophobic nature.As a result, CDs advantageously improve properties of the guestcompounds such as stability to heat, oxygen or ultraviolet rays andsolubility in water and various solvents. Thus CDs are utilized not onlyin the field of foods but also in the pharmaceutic, cosmetic, pesticideand various other fields.

As mentioned above, CDs are obtained as a mixture of various CDs andmaltooligosaccharides by the action of CGTase on an α-1,4-glucan such asstarch or partial hydrolysate thereof as a mixture of various CDs andmaltooligosccharides but the product yield and yield ratio of the CDsvaries slightly depending on the origin of the CGTase. Severalmicroorganisms are known as sources of CGTases but microorganismsbelonging to Bacillus are known as particularly good producers of aCGTase. For example, the following microorganisms are known: B.macerans(Biochemistry, vol. 7, p 114, 1968 and Agric. Biol. Chem., vol. 38, p387, 1974 and ibid, vol. 38, p 2413, 1974); B.circulans (AmylaseSymposium, vol. 8. p 21, 1973); B.megaterium (Agric. Biol. Chem., vol.38, p 387, 1974 and ibid, vol. 38, p 2413, 1974); B.stearothermophilus(Japanese Patent No. 947335, J. Jap. Soc. Starch Sci., vol. 29, p 7,1982); and alkaliphilic Bacillus sp. (Die Starke, vol. 27, p 410, 1975,Agric. Biol. Chem., vol. 40, p 935, 1976 and ibid, vol. 40, p 1785,1976).

Aside from microorganisms belonging to Bacillus, Klebsiella pneumoniae(Arch. Microbiology, vol. 111, p 271, 1977, Carbohydr. Res., vol. 78, p133, 1980 and ibid, vol. 78, p 147, 1980) is reported to produce aCGTase. The CGTases produced by various micoorganisms are classifiedinto α-CD and β-CD-forming types on the basis of the initial reactionproduct produced from starch. B.macerans and K.pneumoniae produceα-CD-forming type and B.circulans, B.megaterium and alkaliphilicBacillus sp. produce β-CD-forming type. Further, the CDs content of areaction product produced by a CGTase of B.stearotheromohilus fromstarch is as follows: β-CD>α-CD>Γ-CD. However, this CGTase is properlyα-CD type because the initial reaction product of this CGTase is α-CD(Japanese Patent No. 947335 and J. Jap. Soc. Starch Sci., vol. 29, p 13,1982).

A CGTase of Γ-CD producing type was recently found but its Γ-CDproductivity is quite low, making it inappropriate for practical use(Denpun Kagaku, vol. 33, p 137, 1986 and Japanese Patent Disclosure No.61-274680).

Proper use of a CGTase according to purpose is advantageous sinceCGTases from different sources exhibit different action depending on pHand temperature, as well as different product yields and yield ratios ofCDs from starch. However, a CGTase with a suitable optimum temperatureand pH or better stability to temperature and pH is easier to useindustrially. In general, it is preferred that the optimum temperatureof an enzyme used for production of a starch sugar such as amylase bebetween 65° and 70° C. and that the optimum pH thereof be in the weakacid range of from 4.5 to 6.5. One reason for setting the optimumtemperature at 65° to 70° C. is to prevent microorganism pollution ofthe reaction solution. Microoganism pollution makes it necessary to uselarge amounts of alkaline agents such as sodium hydroxide to compensatefor pH drop of the reaction solution which results from the pollution,and also requires purification by, for example, deionizing anddecoloring, which makes the process expensive and difficult. Further,use of CGTases having higher optimum temperature and stable temperatureis not economical because it requires more energy to maintain thetemperature of the reaction vessel and inactivate the enzymes.

Moreover, when CGTases having an optimum pH in the alkaline range areused, an isomeric reaction occurring simultaneously with the enzymereaction and colorization reduce the product yield and make thepurification operation complicated. Therefore use of such a CGTase isnot economical.

As described above, CGTases useful for industrial processes have toexhibit not only high yield of CDs from starch but also suitable optimumtemperature, optimum pH and temperature stability for practical use.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a β-CD type CGTasecapable of producing CDs from starch cheaply and with high productivity.

Another object of the present invention is to provide a β-CD type CGTasewhich has an optimum temperature of about 65° C., at which pollutionwith microorganisms during reaction can be prevented, which can beinactivated at about 80° C. and which has an optimum pH in the weak acidrange.

A further object of the present invention is to provide a process forpreparation of a novel CGTase having the above properties.

Other objects of the present invention will be apparent from thefollowing detailed explanation of the invention.

The aforementioned objects of the invention can be accomplished byproviding a CGTase having the following physical properties: (a) Action:Cuts α-1,4-glucopyranoside bonds of an α-1,4-glucan such as starch andglycogen, or partial hydrolysate thereof and transfers it to formcyclodextrins and produces β-cyclodextrin as the initial reactionproduct of its transferring;

(b) Substrate specificity: Reacts with a maltooligosaccharide havingα-1,4-glucopyranosidic linkages with a chain length not less than thatof maltotriose to form various maltooligosaccharides with variousmolecular weight and cyclodextrins by intermolecular disproportionatingreaction between substrates;

(c) Optimum pH: About 6.5

(d) Stable pH: Stable at pH 6 to 9 at 40° C. for 2 hours;

(e) Optimum temperature: About 65° C.;

(f) Inactivation condition: Completely inactivated by treatment at pH of4.5 and 11.5 at a temperature of 40° C. for 2 hours and by treatment atpH of 6 at 75° C. for 15 minutes;

(g) Heat stability: Stable up to 50° C. under the condition of pH 6 for15 minutes and residual activity at 60° C. and 70° C. is 95% and 20%respectively under the same condition;

(h) Inhibition: Inhibited by mercury (II) or copper(II);

(i) Activation and stabilization: Stabilized by calcium;

(j) Molecular weight (SDS-polyacrylamide gel electrophoresis):36,000±1,000;

(k) Isoelectric point (chromatofocusing): 4.8±0.1.

BRIEF EXPLANATION OF THE FIGURES

FIG. 1 shows time-course change of α, β, Γ and total CD when the CGTaseof the present invention is used.

FIGS. 2 and 4 respectively show the pH effect and temperature effect onthe CGTase activity.

FIGS. 3 and 5 show the profiles of pH- and thermal-stabilities of theenzyme.

FIG. 6 shows the effect of Ca²⁺ concentration on the thermal stabilityof the CGTase of the present invention.

FIG. 7 shows the calculation of molecular weight of the CGTase of thepresent invention by SDS-PAGE.

FIG. 8 is a figure showing the isoelectric point of the CGTase of thepresent invention to be 4.8±0.1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The CGTase of the present invention can be produced by cultivating aCGTase producing microorganism belonging to Bacillus coagulans, which isa moderate thermophile obtained from soil, and collecting the CGTaseproduced extracellularly into the culture fluid.

The CGTase producing microorganism used in the present invention whichhas been discovered and isolated from soil by the inventors of thisinvention was identified as Bacillus coagulans from the facts that it isaerobic, Gram-positive, spore-forming rods, motile and peritrichousflagella. Taxonomical studies of the isolated strain was performed withreference to Bergey's Manual of Systematic Bacteriology, vol. 1published by Williams and Wilkius, Baltimore/London and The GenusBacillus, published by the U.S. Department of Agriculture. Further theisolated strain has been identified as Bacillus coagulans from the factthat it does not grow at 65° C. but grows at a temperature of from 30°to 60° C. and in the presence of 0.02% of sodium azide, does not produceammonia from urea, and produces dihydroxyacetone.

Japanese Patent No. 947335 discloses that a CGTase producingmicroorganism (FERM P-2219) belonging to B.stearothermophilus grows at60° C. and does not grow at 65° C. However, the isolated strain(YH-1306) of the present invention grows in the presence of 3% sodiumchloride and cannot utilize inorganic nitrogen compounds, the shape ofthe sporangium is different from that of FERM P-2219 and it can beconcluded that the isolated bacterial strain of the present invention isa novel microorganism producing CGTase. Further, as described below, theenzymatic properties and substrate specificity of the CGTase produced bythe strain (YH-1306) are different from those described in JapanesePatent No. 947335 and therefore the strain (YH-1306) is considered to benovel. The isolated strain (YH-1306) does not grow under alkaliphilicconditions and does not assimilate citric acid and is thereforeobviously different from B.megaterium and B.circulans. In conclusion,the strain (YH-1306) of the present invention is identified as amicroorganism belonging to B.coaqulans. Taxonomical properties of thestrain (YH-1306) are listed in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Taxonomical properties of YH-1306                                             ______________________________________                                        1.  Morphological characteristics                                                 Gram-positive Bacillus (0.7 to 1.0 micron × 2 to 4                      microns) having motility and peritrichous flagella. The                       sporangium is not expanded or only slightly expanded and the                  spores (0.9 to 1.0 micron × 1.5 to 2.0 microns) are                     ellipsoidal and located at the end or near the end of the                     sporangium.                                                               2.  Growth in various cultures                                                    Nutrient agar plate culture: Forms yellowish round                            colonies. Surface elevations are capitate.                                    Nutrient agar slant culture: Grows in a spreading form.                       Nutrient gelatin agar stab culture: Gelatin is not                            liquidized.                                                                   Litmus milk: Produces acid without coagulation.                               Broth liquid culture:                                                          0.5% NaCl: Grows, with sediment                                                3% NaCl: Grows slightly                                                       5% NaCl: Does not grow                                                      0.02% azide: Grows slightly                                               3.  Growth pH and temperature                                                     Growth pH: 5.8 to 7.5 (Optimum pH: 6.5 to 7.0)                                Growth temperature: 30 to 60° C.                                       (Optimum temperature: 45 to 50° C.)                                4.  Biochemical properties                                                    Reduction of nitrate  -                                                       Denitrification       -                                                       Methylred test        -                                                       VP test               -                                                       Production of acid in VP broth                                                                      +                                                       Production of ammonia from urea                                                                     -                                                       Production of indole  -                                                       Production of hydrogen sulfide                                                                      -                                                       Production of dihydroxyacetone                                                                      +                                                       Decomposition of tyrosine                                                                           -                                                       Assimilation of citric acid                                                                         -                                                       Decomposition of casein                                                                             +                                                       Hydrolyzation of starch                                                                             +                                                       Liquefaction of gelatin                                                                             +                                                       Utilization of inorganic                                                                            -                                                       nitrogen compounds                                                            Production of pigment(s): Yellow pigment is produced                              Urease test           +                                                       Oxidase test          -                                                       Catalase test         +                                                       Production of gas from glucose                                                                      -                                                       Production of acid from sugar                                                 Glucose               +                                                       Arabinose             +                                                       Lactic acid           -                                                       Mannitol              +                                                   5.  Other properties                                                              OF test: Oxidative                                                        Behavior in oxygen: Aerobic (grows only slightly in an                        anaerobic condition)                                                          GC content: 46 ± 1% (HPLC)                                                 Resistivity to lysozyme                                                                             -                                                       ______________________________________                                         +: positive or grows well                                                     -: negative or does not grow                                             

The strain (YH-1306) used in the present invention was deposited withthe Fermentation Research Institute (FRI) in accordance with theBudapest Treaty under the accession number of FERM BP-2258.

The novel CGTase of the present invention will now be explained indetail. The CGTase producing microorganism belonging to B.coagulans isinoculated to a culture whose pH is regulated to 5.8 to 7.5, preferablyfrom 6.5 to 7.0, and then the inoculted culture is aerobicallycultivated at a temperature between 30° and 60° C., preferably between45° and 50° C. for from 30 to 80 hours to produce and accumulate theCGTase in the cultivation fluid as an extracelluar enzyme.

Various materials which are known and cheaply available can be appliedto the culture used in the present invention. Examples of nitrogensources include corn steep liquor, polypeptone, soybean meal, bran, meatextract, yeast extract and amino acid solution. Examples of carbonsources include corn syrup, maltose, various starchs, soluble starch,liquefied starch, dextrin and pullulan.

In addition to the nitrogen and carbon sources, various salts such asinorganic salts (for example magnesium salt, potassium salt, phosphate,ferric salt) and various vitamins may be optionally added to theculture.

An example of a culture suitable for the above cultivation is a liquidculture containing 5% corn steep liquor, 0.1% K₂ HPO₄, 0.02% MgSO₄.7H₂ Oand 0.005% CaCl₂.2H₂ O.

The CGTase producing microorganism (FERM BP-2258) used in the presentinvention produces an extracellular enzyme. The extracellular enzymeaccumulated in the culture supernatant is subjected to centrifugalseparation to remove microbial cells and obtain a crude enzyme. Use ofthe resulting crude enzyme without purification is economical. However,the crude enzyme may be purified and this purification can be conductedby, for example, salting out with ammonium sulfate, solventprecipitation with ethanol, acetone or iso-propanol, ultrafiltration orgeneral enzyme purification using an ion exchange resin.

A preferred example of a method for preparation of the pure CGTase ofthe present invention will be explained.

A moderate thermophile Bacillus coagulans (YH-1306) is inoculated to amedium containing 5% (w/v) corn steep liquor, 0.1% K₂ PO₄, 0.02%MgSO₄.7H₂ O and 0.005% CaCl₂.2H₂ O and then aerobically cultivated at45° C. for 72 hours under the following conditions: aeration rate of 1v.v.m. and agitation at 250 r.p.m. The resulting cultivation solution issubjected to a continuous centrifugal separation at 10,000 r.p.m. at atemperature of 4° C. to remove bacterial cells and obtain a supernatant(crude enzyme solution). Then solid ammonium sulfate is added to thecrude enzyme solution to saturate up to about 20%. The solution ispassed through a corn starch layer to adsorb the emzyme from thesolution and the enzyme is then extracted with an excess volume of 40 mMdisodium hydrogen phoshate solution. The resulting extract solution isconcentrated by use of an ultrafiltration membrane (average fractionmolecular weight: 10,000) and then the concentrated solution issubjected to dialysis against a 10 mM acetate buffer solution (pH 6.0)containing 1 mM CaCl₂ at 4° C. overnight. The precipitates formed duringthe dialysis are removed by centrifugation. The resulting supernatant isadsorbed on a DEAE-Toyopearl 650M column equilibrated with, for examplethe 10 mM acetate buffer solution and the enzyme is eluted with asimilar buffer solution containing 0 to 0.7M NaCl by the linear gradientmethod. The eluted active fractions are collected and concentrated usingthe above-mentioned ultrafiltration membrane. Then the concentratedfractions are loaded onto a Sephacryl S-200 column equilibrated with thesame buffer solution containing 0.1M NaCl and subjected to eluation withthe buffer solution.

The eluted active fractions are collected and concentrated by theabove-mentioned ultrafiltration membrane. The concentrated fractions aresubjected to high performance liquid chromatography using a ShodexWS-2003 column to collect active fractions. The resulting purifiedenzyme is analyzed by polyacrylamide gel disc electrophoresis (PAGE, gelconcentration: 7.5%) and the analysis shows that the purified enzyme ishomogeneous and the yield of enzyme activity by this purification isabout 25%.

The β-CD-forming activity can be determined by the method of Kaneko etal (J. Jpm. Soc. Starch Sci., vol. 34, p 45-48, 1987) as follows. Thereaction mixture containing 1.0 ml of 4% (w/v) soluble starch solutionin 0.1M acetate buffer solution (pH 6.0) and 0.1 ml of an enzymesolution is incubated at 60° C. for 20 minutes, whereafter the reactionis stopped by addition of 3.5 ml of 30 mM NaOH solution. Then 0.5 ml of0.02% (w/v) phenolphthalein in 5 mM Na₂ CO₃ solution is added to thereaction mixture and the color of the resulting solution after keepingfor about 15 minutes at room temperature is measured by a photoelectriccolorimeter (Klett-Summerson type) using a filter No. 54 (550 nm) with1.0 ml of 0.5 mg/ml β-CD solution, as a standard, which issimulteneously treated with the phenolphthalein solution. A unit of theenzyme activity is defined as follows: amount of the enzyme required toform 1 mg β-CD per 1 minute under the condition described above.

The physical and enzymological properties of the CGTase obtained by themethod of the present invention are compared with those of CGTasesobtained from known bacteria and shown in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Comparison of enzymological properties                                        __________________________________________________________________________            Present                                                               Properties                                                                            Invention   ABC-38      ABC-40                                        __________________________________________________________________________    Origin  B. coagulans                                                                              B. megaterium                                                                             alkalophilic                                                                  Bacillus                                      Optimum 6.5         5 to 7.5    acidic CGTase                                 pH                  peak at 5.5 (A.C.): 4.5                                                       shoulder at 7.5                                                                           neutral CGTase                                                                (N.C.): 7                                     Optimum 65° C.                                                                             55° C.                                                                             A.C.: 45° C.                           temp.                           N.C.: 50° C.                           Temp.   15 minutes  10 minutes  30 minutes                                    Stability                                                                             treatment   treatment   treatment                                     (residual                                                                             60° C.                                                                       93%   55° C.                                                                      100%       A.C.                                                                              N.C.                                  activity)                                                                             65° C.                                                                       55%   60° C.                                                                       25%   40° C.                                                                     100%                                                                              100%                                          70° C.                                                                       20%   65° C.                                                                       2%    50° C.                                                                      25%                                                                              100%                                          75° C.                                                                        0%               60° C.                                                                      0% 100%                                                                  70° C.                                                                      0%  95%                                  pH      40° C., 2 hrs                                                                      40° C., 2 hrs                                                                      60° C., 30 mins                        Stability                                                                             treatment   treatment   treatment                                     (residual                                                                             pH 4.5                                                                                0%  pH 5.7                                                                             7%         A.C.                                                                              N.C.                                  activity)                                                                             5.0   65%   6.0  30-42% pH 5                                                                               5%  20%                                          6-9   100%  7-10 100%   6   90% 100%                                          10    90%   11   67-75% 7-9 100%                                                                              100%                                                                  10  10% 100%                                  Molecular                                                                             36,000 ± 1000                                                                          66,000      85,000-88,000                                 Weight                                                                        Isoelectric                                                                           4.8 ± 0.1                                                                              fraction    5.4                                           point               1: 6.07                                                                       2: 6.80                                                   Initial product                                                                       β-CD   β-CD   β-CD                                     from starch                                                                   Final CDs                                                                     CD > Γa.-CD > α                                                   CD > Γa.-CD > α                                                   CD > Γa.-CD > α                                                   component                                                                     __________________________________________________________________________                        JP Patent No.                                                         Properties                                                                            947335      ABC-38                                        __________________________________________________________________________                Origin  B. stearothermophilus                                                                     B. macerans                                                                   (IFO 3490)                                                Optimum 3.5 to 8.5  5.2 to 5.7                                                pH      peaks at 5.5 and 7.5                                                  Optimum 75° C.                                                                             55° C.                                             temp.                                                                         Temp.   15 minutes  10 minutes                                                Stability                                                                             treatment   treatment                                                 (residual                                                                              60° C.                                                                      100%  55° C.                                                                       100%                                                activity)                                                                             70° C.                                                                       90%   60° C.                                                                       95%                                                         75° C.                                                                       55%   65° C.                                                                       70%                                                         80° C.                                                                       10%   70° C.                                                                       10%                                                 pH      40° C., 2 hrs                                                                      40° C., 2 hrs                                      Stability                                                                             treatment   treatment                                                 (residual                                                                             pH 4.5                                                                              10%   pH 5   0%                                                 activity)                                                                             5.0   80%   6     70%                                                         6-8   100%  7     75%                                                                     8     95%                                                                     9-10  100%                                                                    11    90%                                                 Molecular                                                                             70,000 ± 40,000                                                                        65,000                                                    Weight                                                                        Isoelectric                                                                           4.45        4.6                                                       point                                                                         Initial product                                                                       α-CD  α-CD                                                from starch                                                                   Final CDs                                                                     CD > Γa.-CD > α                                                   CD > Γa.-CD > α                                                   component                                                         __________________________________________________________________________     ABC-38: Agric. Biol. Chem., vol. 38, 387, 1974 and ibid, vol. 38, 2413,       1974                                                                          ABC-40: Agric. Biol. Chem., vol. 40, 935, 1976 and ibid, vol. 40, 1785,       1976                                                                     

As shown in Table 2, the CGTase of the present invention is differentfrom CGTases of B.stearothermphilus and B.macerans in the initialproduct from starch. Further the CGTase of the present invention isdifferent from the enzyme of alkalophilic Bacillus sp. in the CDscomposition of the final product.

Although the initial product and the CDs composition of the finalproduct of the CGTase of the present invention are the same as those ofthe CGTase of B.megaterium, they are different in profiles of pH andtemperature on activity and stability. From the industrial viewpoint,use of the enzyme of B.macerans is preferred for production of α-CD anduse of an enzyme of alkalophilic Bacillus sp. is preferred forproduction of β-CD, but both of these enzymes have disadvantages inoptimum temperature and temperature stability.

Use of CGTases B.mecaterium and B.stearothermophilus is suitable forproduction of ∂, β and Γ-CD in good proportion. However, the CGTase ofB.meqaterium has disadvantages in optimum temperature and temperaturestability and pollution of the reaction mixture with bacteria cannot besufficiently prevented. The CGTase of B.stearothermophilus has highoptimum temperature and exhibits superior temperature stability.

The optimum temperature and temperature stability of an enzyme aregeneraly measured in the absence of a substrate or at a low substrateconcentration but reaction is practically conducted using a highconcentration of substrate such as 10 to 20% (w/v) from an economicalviewpoint. In the solution containing a substrate in a highconcentration, the enzyme is generaly stabilized by the protectingaction of the substrate. Similarly, the CGTase is stabilized in asubstrate solution and exhibits higher temperature stability than in asulution without the substrate. CDs obtained by reaction of a CGTasewith starch are generaly purified by crystalization, membrane separationor chromatography using an ion exchange resin or a gel filtration resinbut after reaction of the CGTase, a fair amount of high molecular weightdextrins and maltooligosaccharides remain in the reaction solution dueto the substrate specificity of the CGTase. These remaining componentsactually make impossible operations such as deionization and filtrationand therefore the reaction solution is subjected to action of amylolyticenzymes such as α-amylase, β-amylase, glucoamylase and α-1,6-glucosidaseto hydrolyze and decompose the high molecular weight dextrins andmaltooligosaccharides and reduce the viscosity of the reaction solution.When the CGTase used to the CDs production reaction is still activeafter the reaction, as mentioned above, the produced CDs are hydrolyzedby coupling and disproportionating actions of the CGTase during thesaccharification step to reduce the viscosity of the solution. For theabove reasons, the CGTase has to be completely inactivated using beforevarious amylolytic enzymes for the re-saccharification of theoligosaccharides and dextrins aside from CDs. Use of acids and alkalineagents is not economical for the following reasons: Undesirable sidereactions such as hydrolysis and isomerization occur and much energy andlabor are necessary for purification such as deionization anddecoloration because pH should be re-adjusted with alkaline agents oracids at the optimum pH of the enzyme used for resaccharification priorto the re-saccharification. Thus heat inactivation is generaly used forinactivation of CGTases. However, the heat inactivation is noteconomical for inactivation of the CGTase of B.Stearothermophilusbecause it has high temperature stability. Under the circumstances, useof the CGTase of the present invention is quite economical because ithas not only an optimum temperature suitable for prevention of microbialpollution of a reaction vessel and temperature stability suitable forheat inactivation but also an optimum pH similar to those of variousamylases used for re-saccharification of dextrins and oligosaccharides.

According to a method of the present invention, the CGTase useful forpreparation of CDs from starch can chaeply be provided in large amounts.Further carbohydrates containing α, β and Γ-CDs in good proportion canbe prepared by the CGTase in large scale.

EXAMPLES

The novel CGTase of the present invention and a method for preparationthereof will now be explained referring to following Examples. However,the scope of the present invention is not limited by the Examples.

EXAMPLE 1

A moderate thermophile Bacillus YH-1306 (FERM BP-2258) strain wasinoculated to 300 ml of a medium (pH 6.8) containing soluble starch 1%,polypeptone 1.5%, yeast extract 0.5%, K₂ HPO₄ 0.1%, MgSO₄.7H₂ O 0.02%and CaCl₂.2H₂ O in a two liters conical flask with flutings andcultivated at 45° C., 200 r.p.m. for 20 hours to form a seed solution.The resulting seed solution 290 ml was added to 15 liters of a mediumhaving the same composition as the medium used above in a 30 liters jarfermentor and aerobically cultivated at 45° C. and at an aeration rateof 1.5 v.v.m. for 48 hours under agitation at 250 r.p.m. Aftercultivation, bactrial cells were removed by continuous centrifugation(12,000 g, 4° C.) and the resulting supernatant was subjected toconcentration by use of an ultrafiltration (average molecular weight:10,000) to make the concentration about ten times as that of thesupernatant and obtain about 1.5 liters of a crude enzyme solutionhaving 180 units/ml of the CGTase.

Then solid ammonium sulfate is added to the crude enzyme solution tosaturate up to 20%. The solution is passed through a corn starch layerto adsorb the emzyme from the solution and the enzyme is then extractedwith an excess volume of 40 mM disodium hydrogen phoshate solution. Theresulting extract solution is concentrated by use of an ultrafiltrationmembrane (average fraction molecular weight: 10,000) and then theconcentrated solution is subjected to dialysis against a 10 mM acetatebuffer solution (pH 6.0) containing 1 mM CaCl₂ at 4° C. overnight. Theprecipitates formed during the dialysis are removed by centrifugation.The resulting supernatant is adsorbed on a DEAE-Toyopearl 650M columnequilibrated with the 10 mM acetate buffer solution and the enzyme iseluted with a similar buffer solution containing 0 to 0.7M NaCl by thelinear gradient method. The eluted active fractions are collected andconcentrated using the above-mentioned ultrafiltration membrane.

The resulting crude enzyme solution was loaded onto a Sephacryl S-200column equilibrated with a 10 mM acetate buffer solution (pH 6.0)containing 0.1M sodium chloride and eluation was conducted with the samebuffer solution. Eluted active fractions were collected and concentratedusing the ultrafiltration membrane used above. The concentrated solutionwas subjected to high performance liquid chromatography using a ShodexWS-2003 column to collect active fractions. The resulting purifiedenzyme is analyzed by polyacrylamide gel disc electrophoresis (gelconcentration: 7.5%) and the analysis showed that the purified enzymehad single component and the active yield was about 25%.

EXAMPLE 2

(a) 100 ml of 13% (w/v) potato starch suspension (pH 6.5) containing 3mM CaCl₂.2H₂ O was liquefied using a bacterial PG,24 liquefying typeα-amylase (manufactured by Daiwa Kasei K.K., Chrystase L-1) at 90° to92° C. and immediately heated at 125° C. for 30 minutes to obtain astarch liquefied solution with 1.2 of D.E. (Dextrose Equivalent: ratioof total solid to direct reducing sugar). Then the purified CGTaseobtained in Example 1 was added to the starch liquefied solution in anamount of three units per one gram of the starch liquefied solution andreacted at 70° C. and pH 6.5. This reaction mixture was sampledtime-coursely and CDs in the sampled reaction mixture were measured byhigh performance liquid chromatography (HPLC) using a HPX-42A columnmanufactured by Bio-Rad Lab. Results are shown in FIG. 1.

(b) Specificity

55 mg of an oligosaccharide (maltose, maltotriose or maltotetraose) and1 mM CaCl₂.2H₂ O was dissolved in water 1.0 ml. To the resultingsolution, five units of the purified CGTase of the present invention wasadded and the reaction was conducted at 65° C. for 24 hours. Theobtained reaction mixture was subjected to measurement by HLPC andresults are listed in Table 3.

                                      TABLE 3                                     __________________________________________________________________________    Action on various oligosaccharides                                            Reaction products (%)                                                         Sub                                                                              G.sub.1                                                                          G.sub.2                                                                          G.sub.3                                                                          G.sub.4                                                                          G.sub.5                                                                          G.sub.6                                                                          others                                                                            α-CD                                                                        β-CD                                                                         Γ-CD                                   __________________________________________________________________________    G.sub.2                                                                           0.3                                                                             99.6                                                                              0.1                                                                             ND ND ND ND  ND  ND  ND                                           G.sub.3                                                                          16.9                                                                             20.4                                                                             16.8                                                                             12.9                                                                              9.8                                                                              6.7                                                                             12.8                                                                              1.3 1.9 0.5                                          G.sub.4                                                                           9.0                                                                             15.2                                                                             14.4                                                                             13.0                                                                             10.7                                                                             11.6                                                                             19.7                                                                              2.6 3.0 0.8                                          __________________________________________________________________________     Sub: substrate, G.sub.1 : glucose G.sub.2 : maltose, G.sub.3 :                maltotriose, G.sub.4 : maltotetraose, G.sub.5 : maltopentaose, G.sub.6 :      maltohexaose, ND: not detected                                           

(c) Optimum pH

The purified CGTase of the present invention was subjected to dialysiswith 1 mM EDTA solution overnight and then with water overnight. Theresulting CGTase solution (0.1 ml) deluted suitably with deionized waterwas mixed with 1 ml of 4% (w/v) soluble starch in 0.1M acetic acidbuffer solutions (pH 4.2, 5.0, 5.5 and 6.0) or 0.1M phosphate buffersolutions (pH 6.0, 6.5. 7.0 and 8.0) and the activity of the CGTase wasmeasured as mentioned above. The relative activity at pH 6.5 was made100% and the pH dependence of the relative activity of the CGTase isshown in FIG. 2.

(d) Stable pH

The dialyzed CGTase of the present invention obtained by the sameprocedures as those of (c) was heat treated at 40° C. for 2 hours atvarious pH values (4 to 10). Then the residual activity of the CGTasewas measured. The activity before heating at pH 6.5 was made 100% andthe pH dependence of the residual activity is shown in FIG. 3. In theabove procedures, following buffer solutions were used: acetate buffersolutions (pH 4 to 6), phosphate buffer solutions (6 to 8) andglycine-NaOH-NaCl buffer solutions (pH 9 and 10).

(e) Optimum temperature for action

The activity of the dialyzed CGTase of the present invention obtained bythe same procedures as those of (c) above was measured at varioustemperatures. The temperature dependence of the relative activity of theCGTase (relative activity at 65° C.: 100%) is shown in FIG. 4.

(f) Inactivation and (g) temperature stability

The dialyzed CGTase of the present invention obtained by the sameprocedures as those of (c) was treated at various temperatures (40° to80° C.) for 15 minutes in the presence or in the absence of 5 mMCaCl₂.2H₂ O and then the residual activity was measured. Results areshown in FIG. 5.

(h) Inhibition

A chloride form of various metals was added to a solution containing thedialyzed CGTase of the present invention obtained by the same proceduresas those of (c) to make the concentration thereof 1 mM (mercurry: 0.1mM) and then activity of the CGTase was measured. Results are shown inTable 4 as the relative activity (the reltive activity of the CGTase inthe absence of metal was made 100%).

                  TABLE 4                                                         ______________________________________                                        Effect of metal salts on enzyme activity                                                Relative              Relative                                      Metal salt                                                                              activity    Metal salt                                                                              activity                                      ______________________________________                                        control   100         Cu.sup.2+ 36.0                                          Mn.sup.2+ 79.3        Zn.sup.2+ 62.6                                          Pb.sup.2+ 91.8        Co.sup.2+ 75.9                                          Mg.sup.2+ 80.3        Hg.sup.2+  9.9                                          Ni.sup.2+ 93.6        Fe.sup.2+ 92.6                                          Cd.sup.2+ 94.8        Fe.sup.3+ 98.8                                          ______________________________________                                    

(i) Stabilization

Various CaCl₂ solutions (CaCl₂ concentration: 0 to 3 mM) were added tosolutions of the dialyzed CGTase of the present invention obtained bythe same procedures as those of (c) and maintained at 75° for 15minutes. Then the residual activity of the CGTase was measured and shownin FIG. 6 as the relative activity (the relative activity in thepresence of 3 mM CaCl₂ was made 100%).

(j) Molecular weight

The molecular weight of the CGTase of the present invention was measuredby SDS-polyacrylamide gel electrophoresis (SDS-PAGE) described in K.Weber and M. Osborn, J. Biol. Chem., vol. 244, p 4406, 1969 (FIG. 7).The molecular weight of the present invention was 36,000±1,000.

(k) Isoelectric point

The isoelectric point of the CGTase of the present invention wasmeasured by the chromatofocusing method using PBE94 gel and Polybuffer74 manufactured by Pharmacia and was 4.8±0.1.

As described above, according to the present invention, the CGTase whichhas an optimum temperature of the enzyme reaction at about 65° C., whichis completely inactivated at about 80° C. and which has an optimum pHsimilar to those of various amylolytic enzymes used forre-saccharification after CDs producing reaction is provided.

The CGTase produces maltooligosaccharides containing β-CD as maincomponents together with an apropriate amount of α-CD and Γ-CD fromstarch. Further the method for preparation of CDs using the CGTase ofthe present invention is economical since no agent is required for themethod, additional treatment such as deionization can be simplified andheat energy can be saved.

Thus the present invention makes possible to produce CDs cheaply inlarge scale and the present invention has industrial significance.

What we claim is:
 1. A method for preparing a cyclomaltodextringlucanotransferase having the following physical properties:(a) optimumpH: about 6.5, (b) optimum temperature: about 65° C., and (c) heatstability: stable up to 50° C. at pH 6 for 15 minutes and havingresidual activity at 60° C. and 70° C., of 95% and 20%, respectively,under the same conditions, comprising the steps of: culturing Bacilluscoagulans FERM BP-2258 and producing said cyclomaltodextringlucanotransferase, and collecting said cyclomaltodextringlucanotransferase from the resulting culture broth.
 2. A method ofclaim 1 wherein the cultivation is conducted aerobically at 30° to 60°C.
 3. A method of claim 1 wherein pH of a culture solution used for thecultivation ranges from 5.8 to 7.5.